Colorimetric determination of calcium in biologic fluids

ABSTRACT

Methylthymol blue dye in colorimetrically analyzing a biologic fluid such as blood serum for calcium content. Analysis can be accomplished in the presence of proteins, phosphates and bilirubin. 8-Quinolinol can be used to bind magnesium without an accompanying adverse effect on the measurement of calcium concentration. Color stable aqueous solutions of the dye are also provided as well as color stability of the calcium-dye complex during analysis.

United States Patent 1 Gindler 1 Aug. 28, 1973 COLORIMETRICDETERMINATION OF CALCIUM IN BIOLOGIC FLUIDS [75] Inventor: E. MelvinGlndler, Rockford, Ill.

[73] Assignee: Pierce Chemical Company, 7

Rockford, Ill.

[22] Filed: Jan. 5, 1972 [21] Appl. No.: 215,638

[52] U.S. Cl 23/230 B [51] Int. Cl. G0ln 33/16 [58] Field of Search23/230 B, 253 TI;

[56] References Cited UNITED STATES PATENTS 7/1969 Fraguada 23/230 BOTHER PUBLICATIONS Belcher et al., New Methods of Analytical Chemistry,

2nd ed., 1964, Reinhold Pub. Co., pp. 46, 47. relied on. Srivastava etal., Chem. Abstr. 71, 1 19209 H1969).

Chauhan et al., Analytical Biochem. 32, 70-80 (1969) PrimaryExaminer-Morris O. Wolk Assistant Examiner-R. M. Reese Attorney-C.Frederick Leydig, Thomas E. Currier et a1.

[57] ABSTRACT 28 Claims, No Drawings I COLORIMETRIC DETERMINATION OFCALCIUM IN BIOLOGIC FLUIDS BACKGROUND OF THE INVENTION The presentinvention relates to the analysis of biologic fluids and, moreparticularly, to the colorimetric determination of the calcium contentin blood serum and the like.

Colorimetric methods are widely used for determining the concentrationof various elements such as calcium and magnesium in biologic fluidssuch as blood. The methods usually involve adding a reagent to the fluidwhich forms a colored complex with the element to be detected. Thecomplex absorbs light at a characteristic wave length. By exposing asample of the fluid containing the complexed reagent to a light sourceof the characteristic wave length and thereafter measuring the degree oflight absorption, the content of the element in the biologic fluid canbe simply determined from a calibration graph constructed fromabsorbance measurements made on standard reagent dye solutionscontaining known concentrations of the element. Reagents formingcomplexes with calcium which have been used in determining the calciumconcentration in biologic fluids include o-cresolphthalein complexone,sodium alizarinsulfonate, and sodium rhodizonate.

One of the problems encountered in determining calcium concentration inbiologic fluids by colorimetric techniques is the effect on theabsorption spectrum of ingredients such as serum proteins, phosphates,magnesium and bilirubin normally present in such fluids. Serum proteinsand phosphates tend to bind calcium, thus lowering the quantity ofcalcium complexed with the reagent dye. The colorimetrically determinedapparent quantity of calcium is then lower than that actually present.Magnesium complexes with many dyes give an absorption spectrum similarto the calcium complex. The colorimetric determination then gives aresult based on the combined concentrations of calcium and magnesium.Bilirubin itself, in the concentrations present in normal or abnormal(e.g., jaundiced) serum, absorbs light at a wave length close to that ofmaximum absorbance of many calcium-dye complexes. Thus, the presence ofbilirubin can interfere with the accurate colorimetric determination forcalcium.

in order to overcome the problems associated with the presence ofproteins and bilirubin, the biologic fluid can be subjected to dialysisto remove these ingredients prior to the colorimetric determination ofcalcium concentration. This, of course, is time consuming and can leadto added expense as well as introduce potential errors in the subsequentdetermination of calcium concentration. The potentially adverse effectof magnesium present in the fluid can be diminished by adding a reagentto the fluid which has a stronger affinity for complexing magnesium thandoes the reagent dye. 8- Quinolinol has been used for this purpose. Withthis approach, it is necessary that a reagent dye be used which cancompete effectively with S-quinolinol for calcium.

Accordingly, a principal object of the present invention is to providean operationally simple and rapid colorimetric method for accuratelydetermining the calcium concentration in biologic fluids which can beaccomplished in the presence of serum proteins, phosphates, andbilirubin. A closely related object is to provide a method as called forin the principal object wherein the reagent dye used has a very strongaffinity for calcium and permits the use of magnesium complexing agentswithout a significant adverse effect on the calcium determination.

Other problems associated with colorimetric analysis involve thestability of the reagent solution used in making the colorimetricdetermination and the stability of the colored calcium complexed dye.Stability of the reagent solution is important where it is desired toprepare the working solution in advance of its actual use. Stability ofthe colored dye-calcium complex after formation is important inpermitting an accurate determination of calcium concentration where thebiologic fluid is not analyzed immediately after addition of the reagentdye. In the absence of color stability, drifting of the observedcolorimeter or spectrophotometer reading will occur making the accuratedetermination of calcium concentration difficult.

Consequently, a further object of the present invention resides inproviding a reagent dye solution which can be used in colorimetricallyanalyzing biologic fluids and which is stable for a number of months,thus enabling its preparation and storage prior to actual use.

An additional object resides in providing a biologic fluid containing acolored dye-calcium complex wherein the color of the complex is stablefor several hours after'addition of the reagent dye to the fluid, thusobviating the necessity for immediate analysis of the fluid after dyeaddition.

Other objects and advantages of the present invention will becomeapparent upon reading the following detailed description.

While the invention is susceptible of various modifications andalternative constructions, there will herein be described in detail thepreferred embodiments. It is to be understood, however, that it is notintended to limit the invention to the specific forms disclosed. On thecontrary, it is intended to cover all modifications and alternativeconstructions falling within the spirit and scope of the invention asexpressed in the appended claims.

Briefly stated, one embodiment of the present invention involves the useof methylthymol blue as the reagent dye for the colorimetricdetermination of calcium concentration in biologic fluids. The use ofthis dye is accompanied by a combination of unexpected and significantadvantages. Methylthymol blue dye has been found to be particularlyefficient in complexing with substantially all of the calcium present inthe .fluid. As a result, significant concentrations of proteins andphosphates do not adversely affect the accuracy of the calciumdetermination and dialysis to remove proteins is not needed. Similarly,the removal of bilirubin is not necessary since the characteristicmaximum absorption wave length for the colored calcium-dye complex issubstantially different than that of bilirubin. The use of this reagentdye also permits the addition of magnesium complexing agents to thefluid without the associated risk that calcium will be preferentiallycomplexed therewith and detract from the accuracy of the measurement forcalcium. By using methylthymol blue in accordance with the presentinvention, colorimetric analysis of blood serum and the like for calciumcan be accomplished within about five minutes after the biologic fluidis obtained. Furthermore, the sensitivity of the process is sufficientto permit ultramicro application.

In accordance with a further aspect of the present invention, it hasbeen found that a stable aqueous solution of methylthymol blue can beprepared by simply making the solution acidic and including therein amicelle forming protective colloid. The acidic solution is stable for atleast 1 year and, accordingly, can be prepared and then stored forsubsequent use.

While, as indicated above, methylthymol blue is useful for thecolorimetric determination of calcium concentration, it has been notedthat some fading of the color of the dye-calcium complex occurs withpassage of time. In accordance with still a further feature of thepresent invention, it has been discovered that by adding a reducingagent to the biologic fluid containing the colored calcium-dye complex,or alternatively to a prepared aqueous reagent dye solution, this fadingcan be substantially delayed. The result is that the color of the sampleto be analyzed is stable for as long as several hours and the necessityfor immediate analysis is avoided.

Methylthymol blue is a well known chelating or metal-binding dye. Thedye is commercially available generally as the pentasodium salt and forthe purposes of the present invention should be used in a very pureform. ln particular, no calcium or other multivalent metal impuritiesshould be present. The amount of dye employed in accomplishing thepresent process should be at least equivalent to that necessary tocomplex with all of the calcium in the fluid to be analyzed. So long asmagnesium is independently complexed with another reagent, the use ofexcess quantities of the dye is accompanied by no undesirable efi'ectsother than those of economics through an increasingly intense lightsource is required with increasing quantities of dye in order to allowthe colorimeter or spectrophotometer to be set at zero absorbance whencalcium is absent.

With human blood serum or plasma, the use of about (2.4 x (4.0 X 10) andpreferably (3 X 10") (3 X 10 millimole of methylthymol blue dye per 50microliters of biologic fluid is ordinarily sufficient to permitcolorimetric analysis. Since the uncomplexed dye strongly absorbs lightoutside of a pH range of about 10-13 analysis should be accomplishedwithin this range. To this end, suitable buffers which function in thepH range of 10-13 and do not significantly bind calcium or formprecipitates therewith can be used. Examples of useful buffers includealkyl amines such as hydroxyalkyl amines, e.g., monoethanolamine;aliphatic alkyl amines, e.g., diethylamine, triethylamine; aromaticalkyl amines, e.g., benzylamines, 2- phenylethyl amine; and calcium freecongugate acidbase pairs, e.g. ammonium ion such as from ammoniumchloride, ammonium borate, or ammonium acetate and ammonia.

While the method of the present invention employing methylthymol bluedye has the decided advantage of permitting the direct colorimetricanalysis of biologic fluids without the necessity for removingphosphates, bilirubin or proteins, it is preferred that, in addition tothe dye, a high molecular weight, calcium free micelleforming protectivecolloid also be present during analysis. Several advantages are thoughtto accompany the use of the colloid.

The colloid can hold lipids in solution and thus prevent interference inthe absorption spectrum due to turbidity. In addition to preventingturbidity, it is believed that the use of the protective colloid is alsoimportant in eliminating errors due to the presence of proteins in thebiologic fluid being analyzed. It is known that proteins can bind withdyes (unmetallized or metallized) and thereby influence the colorimetricspectrum obtained for the fluid. However, since the determination ofcalcium concentration is obtained by comparing the observed absorbanceof the biologic fluid sample with the observed absorbance on standardprotein free aqueous solutions containing known calcium concentrations,the spectral influence due to a protein-dye complex is not present inthe measurement on the standard and this can result in erroneousresults. The use of a protective colloid in both the fluid beinganalyzed and the standard is believed to minimize this potential forerror. It appears that the colloid either functions to strongly bind thereagent dye itself and thereby prevent the dye from binding with proteinin the biologic fluid sample or forms a colloid-dye complex in thestandard having an absorbance spectrum similar to the protein-dyespectrum present in the biologic fluid.

Useful protective colloids include polyvinylpyrrolidone and non-ionicsurfactants such as epoxide polymers and copolymers, e.g., polyethyleneand polypropylene oxide, the 9-ethyleneoxide adduct of pnonylphenol;polyvinyl alcohols; carbohydrate polymers; betaines; and high molecularweight anionic and cationic surfactants and polymers such as sodiumdodecyl sulphate and hexadecyl trimethyl ammonium chloride or bromide,as well as mixtures thereof. Based on about microliters of biologicfluid, about 4.5 13.5 milligrams of protective colloid are desirablyemployed.

Concerning magnesium, it is necessary that magnesium either be removedfrom the fluid prior to analysis or 104 be complexed in a mannerwhichprevents it from complexing with the methylthymol blue dye. 8-Quinolinol is a suitable complexing agent which can be used. And, due tothe exceptionally strong calcium binding power of methylthymol blue,8-quinolinol can be used in concentrations sufficient to effectively tieup the magnesium present while not running the risk that calcium will bedrawn away from the dye. With human blood serum or plasma about 3.5 X 1011.2 X 10 and preferably at least about 5 X 10 millimole of thecomplexing agent per 50 microliters of biologic fluid sample can beused.

It has also been indicated previously that a reducing agent is desirablyincluded in the biologic fluid to be analyzed in orderto stabilize thecolor of the calciumdye complex. Useful reducing agents are those whichfunction as antioxidants and include, for example, sodium sulfite andsalts of hydroxylamine such as hydroxylammonium chloride. In order toeffect color stabilization for extended periods on the order of severalhours or more, a molar excess of the reducing agent, based on the dye,of at least several hundred times, and preferably at least about 500times, should be used. The use of such large excesses also results insubstantially the same ionic environment being present in the sample tobe analyzed and in the standard solution used for calibration. In thismanner, any potentially disturbing effects from the ionic atmosphere inthe biologic fluid such as from the presence of proteins can beeliminated.

As indicated, the method of the present invention can be simplypracticed by independently adding methylthymol blue dye and the otherindicated ingredients to a sample of biologic fluid and thereaftercolorimetrically analyzing it for calcium. In order to avoid inducementof protein precipitation by the added reagents, water is desirably alsoadded to the sample. The added water should preferably be at least about40 times, by volume, the amount of biologic fluid sample used.

While the present method can be accomplished by direct addition ofreagents as above indicated, the customary procedure employed byhospitals and the like is to use pre-formulated compositions, generallyaqueous solutions, containing the reagent dye and the other desiredingredients. The pro-formulated compositions are generally referred toas diagnostic kits and are sold by a number of chemical supplycompanies. The principal advantage of diagnostic kits" is thatlaboratory workers such as hospital laboratory personnel do not have tobe concerned with separately adding the various necessary ingredients intheir proper amounts to the samples to be analyzed. The use ofdiagnostic kits speeds the analytical process and reduces the chances ofobtaining incorrect results based on, for example, the use ofcontaminated or improper ingredients.

The problem with preparing a diagnostic kit with methylthymol blue dyeis that the dye has been thought .to be chemically unstable in aqueoussolution. It has now been discovered that this is not so with respect toacidic solutions of the dye containing the aboveindicated protectivecolloid and, preferably, also the magnesium complexing agent. Theseaqueous solutions of methylthymol blue can be stabilized for as long asabout 1 year by maintaining the pH of the solution below about 4. Simplyadding a strong acid such as hydrochloric acid or other strong acidhaving a water so]- uble calcium salt to an aqueous solution ofmethylthymol blue and the other ingredients in an amount to achieve theindicated pH is sufficient to obtain extended stability.

Referring still to the preparation of diagnostic kits for colorimetricanalysis with methylthymol blue, it is desirable that the kit containtwo separate preformulated aqueous compositions. One composition (thedye solution) contains, in aqueous solution, methylthymol blue dye, anon-ionic protective colloid, a magnesium complexing agent, andsufficient acid to stabilize the dye. The other composition (the basesolution) contains, also in aqueous solution, a buffer forming amine anda reducing agent. The following compositions (reported as concentrationsin one liter of distilled water) illustrate useful dye and basesolutions. Dye Solution Ingredient Amount In General PreferredMethylthymol blue 0.15-0.25 millimole 0.2 milli- (sodium salt) molePolyvinylpyrrolidone 3-9 grams 6 grams Hydroc loric Acid (12 M)0.06-0.18 mole 0.12 mole B-Quinolinol 23-75 millimoles 50 millimolesBase Solution Ingredient Amount In General Preferred Sodium Sulphite0.15-0.25 mole .2 mole Monoethanolamine (Densityp: 1.022, 20C) 2.8-4.2moles v 3.7 moles In use, substantially equal volumes (generally about240 milliliters) of the dye and base solution are mixed together priorto making the desired colorimetric calcium determination. About 3milliliters of the mixed dye-base solution are conveniently used per 50microliters of biologic fluid sample. Absorbance is read at about 612 nmwave length using well known colorimetric or spectrophotometrictechniques. A calibration graph can be used to determine the actualconcentration of calcium. The graph can be prepared from colorimetricdeterminations on standard aqueous solutions (50 microliters) containingknown calcium concentrations (from 0 to about 25 milligrams of calciumper .100 milliliters of solution) to which 3 milliliters of thedyebased'solution have been added. As indicated previously, thepossiblility of erroneous measurements due to the presence of protein inthe sample which is not present in the standard or due to ionic strengthdifferences between the sample and standard is substantially eliminatedby the technique described herein. It has been found that with thepresent method Beers law is applicable up to a calcium concentration ofabout 12.5

milligrams per milliliters of sample.

While the present invention has been particularly described with respectto the use of methylthymol blue as the reagent dye, otherphenolsulphonephthaleins containing, as substituents on the phenolrings, a methylenenitrilodiacetic acid group in the 6 and 6- positionscan also be used so long as the calcium complex with the reagent dyeexhibits a maximum absorbance at a wave length substantially removedfrom that of bilirubin which is about 420 nm. To this end, useful dyesalso contain substituents in the 2, 2' and 5, 5'- positions on thephenol rings which serve to shift the maximum absorbance of the calciumdye complex to a wave length of greater than about 590 my" Thesubstituents employed must not themselves be capable of binding calcium.

Useful dyes can be represented as having the following structure:

momcoom. N(CH=COOH)1 -1 5-12 Rr-b r-on R1 4 7-K; 3 c-0 1"i LO:

wherein R, R R and R are spectrum shifting, noncalcium bindingsubstituents. They can be individually selected from alkyl groups (e.g.,methyl, ethyl, propyl, butyl, hexyl, 2-ethylhexyl, decyl, etc.) arylgroups (e.g..

phenyl, halogenated phenyls such as chloro and bromo, l and 2 naphthyl,toluyl, pyridyl, etc.), halogen atoms (e.g., Cl, Br), and oxygen,sulfur, and/or nitrogen containing groups such as ethers (methoxy,ethoxy), thioethers (methylthio ether), sulfoxide (methyl or phenylsulfoxide), sulfonamide, amines (dimethylamino) amide carboxy. Dyeswherein the substituents are lower alkyl groups (e.g., less than aboutcarbon atoms) are thought to be most useful. The most readily availableof these are methylthymol blue (R and R are CH and R and R are (CI-I CH-and methylxylenol blue (R, R,R and R are Cll-I;,-

In addition, one or more of the above substituents may be in the 2", 3",4" and/or 5" positions of the sulfo carboxylic acid-derived ring. Thesulfo carboxylic acid-derived ring may itself be part of a naphthaleneor other isocyclic group or may even be heterocyclic, such as pyridineor quinoxaline. It is even possible that in place of the sulfocarboxylic acid-derived ring there may be a linear group derived from3-sulfo-acrylic acid.

I claim:

1. In a colorimetric process for determining the concentration ofcalcium in a biologic fluid containing proteins, phosphates, andbilirubin comprising 1. adding a reagent dye to the biologic fluid whichforms a colored complex having a characteristic wave length of maximumlight absorption with the calcium present in said fluid while preventingsaid dye from forming colored complexes with other multivalent metalions present in said fluid,

II. exposing said fluid containing the colored calcium-dye complex tolight having the characteristic wave length of maximum absorption forthe complex while maintaining the pH of said fluid at a valve where theuncomplexed dye does not absorb light of the characteristic wave length,

III. measuring the degree of light absorption at said characteristicwave length, and

IV. determining the calcium concentration in said fluid by comparing themeasured degree of light absorption with values of light absorptionobtained from standard aqueous solutions containing the reagent dye andknown calcium concentrations; the improvement wherein said reagent dyeis a phenolsulphonephthalein containing methylenenitrilodiacetic acidgroup substituents in the 6 and 6'- positions on the phenol rings andnon-calcium binding substituents in the 2,2 and 5, 5'positions thereon,said 2, 2' and 5, 5-position substituents shifting the characteristicwave length of maximum light absorption of the calcium complexedphenolsulphonephthalein to greater than about 590 nm thereby permittingthe accurate determination of calcium concentration in the presence ofsubstantial quantities of bilirubin.

2. The process of claim 1 wherein, before colorimetric analysis,8-quinolinol is added to the biologic fluid in an amount sufficient tocomplex with'magnesium present therein.

3. The process of claim 1 wherein, before colorimetric analysis, a highmolecular weight, calcium free micelle forming protective colloid isadded to the biologic fluid to hold lipids in solution;

4. The process of claim 3 wherein the protective colloid is selectedfrom polyvinylpyrrolidone and epoxide copolymers.

5. The process of claim 1 wherein, before colorimetric analysis, areducing agent is added to the biologic fluid to stabilize the color ofthe calcium-dye complex.

6. The process of claim 5 wherein the reducing agent is sodium sulfite.

7. The process of claim I wherein during colorimetric analysis the pH ofthe biologic fluid is maintained at about 10-13 with monoethanol amine.

8. The process of claim 1 wherein the reagent dye is selected frommethylthymol blue and methylxylenol blue.

9. The process of claim 8 wherein, before colorimetric analysis,8-quinolinol is added to the biologic fluid in an amount sufficient tocomplex with magnesium present therein and a high molecular weight,calcium free micelle forming protective colloid is added to the biologicfluid to hold lipids in solution.

10. The process of claim 9 wherein the protective colloid is selectedfrom polyvinylpyrrolidone and epoxide copolymers.

'11. The process of claim 10 wherein a reducing agent is added to thebiologic fluid to stabilize the color of the calcium-dye complex.

12. The process of claim 11 wherein the reducing agent is sodiumsulflte.

13. The process of claim 12 wherein during colorimetric analysis of pHof the biologic fluid is maintained at about 10-13 with monoethanolamine.

14. The process of claim 13 wherein the protective colloid ispolyvinylpyrrolidone.

15. The process of claim 14 wherein the standard aqueous solutionscontaining known calcium concentrations also contain the reagent dye,the polyvinylpyrrolidone protective colloid and the sodium sulfitereducing agent in concentrations substantially the same as those presentin the biologic fluid being analyzed.

16. A stable aqueous dye solution useful in the colorimetricdetermination of calcium in biologic fluids, said solution consistingessentially of (A) water, (B) a reagent dye of phenol-sulphonephthaleincontaining methylenenitrilodiacetic acid group substituents in the 6,6'-positions on the phenol rings and non-calcium binding substituents inthe 2,2 and 5,5,-positions thereon with said 2, 2' and 5, 5'-positionsubstituents serving to shift the characteristic wave length of maximumlight absorption of the dye when complexed with a calcium to greaterthan about 590 nm., (C) a high molecular weight calcium free, micelleforming non-ionic protective colloid, and (D) an acid having a watersoluble calcium salt, the acid being present in an amount sufficient togive a solution pH of less than about 4.

17. The dye solution of claim 16 wherein the protective colloid isselected from polyvinylpyrrolidone, and expoxide copolymers.

18. The dye solution of claim 17 wherein the reagent dye is selectedfrom methylthymol blue and methylxylenol blue and, based on one litterof solution, the protective colloid is present in an amount of about 3-9grams and the reagent dye is present in an amount of about 0.15-0.25millimole.

19. The dye solution of claim 18 wherein the protective colloid ispolyvinylpyrrolidone and the reagent dye is methylthymol blue.

20. The dye solution of claim 19 wherein the acid is hydrochloric acid.

21. The dye solution of claim 16 containing in addition to the recitedingredients a magnesium complexing agent.

22. The dye solution of claim 21 wherein the magnesium complexing agentis 8-quinolinol.

23. The dye solution of claim 22 wherein, per liter of solution,8-quinolinol is present in an amount of about 23-75 millimoles.

24. The dye solution of claim 23 wherein the protective colloid isselected from polyvinylpyrrolidone, and

epoxide copolymers.

25. The dye solution of claim 24 wherein the reagent dye is selectedfrom methylthymol blue and methyl

2. The process of claim 1 wherein, before colorimetric analysis,8-quinolinol is added to the biologic fluid in an amount sufficient tocomplex with magnesium present therein.
 3. ThE process of claim 1wherein, before colorimetric analysis, a high molecular weight, calciumfree micelle forming protective colloid is added to the biologic fluidto hold lipids in solution.
 4. The process of claim 3 wherein theprotective colloid is selected from polyvinylpyrrolidone and epoxidecopolymers.
 5. The process of claim 1 wherein, before colorimetricanalysis, a reducing agent is added to the biologic fluid to stabilizethe color of the calcium-dye complex.
 6. The process of claim 5 whereinthe reducing agent is sodium sulfite.
 7. The process of claim 1 whereinduring colorimetric analysis the pH of the biologic fluid is maintainedat about 10-13 with monoethanol amine.
 8. The process of claim 1 whereinthe reagent dye is selected from methylthymol blue and methylxylenolblue.
 9. The process of claim 8 wherein, before colorimetric analysis,8-quinolinol is added to the biologic fluid in an amount sufficient tocomplex with magnesium present therein and a high molecular weight,calcium free micelle forming protective colloid is added to the biologicfluid to hold lipids in solution.
 10. The process of claim 9 wherein theprotective colloid is selected from polyvinylpyrrolidone and epoxidecopolymers.
 11. The process of claim 10 wherein a reducing agent isadded to the biologic fluid to stabilize the color of the calcium-dyecomplex.
 12. The process of claim 11 wherein the reducing agent issodium sulfite.
 13. The process of claim 12 wherein during colorimetricanalysis of pH of the biologic fluid is maintained at about 10-13 withmonoethanol amine.
 14. The process of claim 13 wherein the protectivecolloid is polyvinylpyrrolidone.
 15. The process of claim 14 wherein thestandard aqueous solutions containing known calcium concentrations alsocontain the reagent dye, the polyvinylpyrrolidone protective colloid andthe sodium sulfite reducing agent in concentrations substantially thesame as those present in the biologic fluid being analyzed.
 16. A stableaqueous dye solution useful in the colorimetric determination of calciumin biologic fluids, said solution consisting essentially of (A) water,(B) a reagent dye of phenol-sulphonephthalein containingmethylenenitrilodiacetic acid group substituents in the 6, 6''-positionson the phenol rings and non-calcium binding substituents in the 2,2''and 5,5,''-positions thereon with said 2, 2'' and 5, 5''-positionsubstituents serving to shift the characteristic wave length of maximumlight absorption of the dye when complexed with a calcium to greaterthan about 590 nm., (C) a high molecular weight calcium free, micelleforming non-ionic protective colloid, and (D) an acid having a watersoluble calcium salt, the acid being present in an amount sufficient togive a solution pH of less than about
 4. 17. The dye solution of claim16 wherein the protective colloid is selected from polyvinylpyrrolidone,and expoxide copolymers.
 18. The dye solution of claim 17 wherein thereagent dye is selected from methylthymol blue and methylxylenol blueand, based on one litter of solution, the protective colloid is presentin an amount of about 3-9 grams and the reagent dye is present in anamount of about 0.15-0.25 millimole.
 19. The dye solution of claim 18wherein the protective colloid is polyvinylpyrrolidone and the reagentdye is methylthymol blue.
 20. The dye solution of claim 19 wherein theacid is hydrochloric acid.
 21. The dye solution of claim 16 containingin addition to the recited ingredients a magnesium complexing agent. 22.The dye solution of claim 21 wherein the magnesium complexing agent is8-quinolinol.
 23. The dye solution of claim 22 wherein, per liter ofsolution, 8-quinolinol is present in an amount of about 23-75millimoles.
 24. The dye solution of claim 23 wherein the protectivecolloid is selected from polyvinylpyrrolidonE, and epoxide copolymers.25. The dye solution of claim 24 wherein the reagent dye is selectedfrom methylthymol blue and methylxylenol blue and, based on one liter ofsolution, the protective colloid is present in an amount of about 3-9grams and the reagent dye is present in an amount of about 0.15-0.25millimole.
 26. The dye solution of claim 25 wherein the protectivecolloid is polyvinylpyrrolidone and the reagent dye is methylthymolblue.
 27. The dye solution of claim 26 wherein the acid is hydrochloricacid.
 28. The process of claim 1 wherein the reagent dye is methylthymolblue.